Bioluminescent Monitoring of Microenvironmental Effects on Multiple Myeloma Engraftment In a Humanized Mouse Model

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1927-1927
Author(s):  
Christina Wu ◽  
Heather Leu ◽  
Wenxue Ma ◽  
Alice Shih ◽  
Dennis Carson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Imaging System ◽  
Ventral View ◽  
Gradient Centrifugation ◽  
The Self ◽  
Post Transplantation ◽  
Self Renewal ◽  
Immunomagnetic Bead

Abstract Abstract 1927 Multiple myeloma (MM) is characterized by clonal proliferation of CD138+ plasma cells in the bone marrow (BM) and remains an incurable disease. Recent identification of a rare population of MM cancer stem cells (MM CSC) is phenotypically similar to memory B cells (CD138- CD34- CD19+) but differs in that they have the self-renewal capacity within the BM and may be responsible for drug resistance. Preclinical testing of novel therapeutic strategies that target MM CSC requires animal models that closely resemble human disease and allow quantitative evaluation of the applied therapy. We have previously reported results on establishing a MM animal model by transplanting MM CSC from autologous mobilized peripheral blood of primary MM patients, transduced with lentiviral luciferase GFP (GLF) and transplanted intrahepatically (IH) into neonatal RAG2/gc double knock-out (RG-KO). Here we evaluate engraftment efficiency in consideration of BM microenvironment by comparing CD45+ human cell engraftment in mice transplanted either IH to neonates or intrafemorally (IF) to gamma-irradiated young adult mice. MM CSC were selected from isolated PBMC after Ficoll gradient centrifugation of fresh BM biopsy from two primary MM patients or from a human MM cell line, H929, followed by immunomagnetic bead depletion of CD34+ and CD138+ cells. The cells were transplanted into RG-KO mice ranging from 53,000 to 10⋀6 cells per mouse either IH or IF. Mice transplanted with GLF-transduced MM CSC were imaged with an in vivo imaging system (IVIS) to detect bioluminescent engraftment. Results showed that bioluminescence signal levels were detected in mice transplanted IF with 53,000 MM CSC per mouse even before 3 weeks by ventral view and as early as 5 weeks by lateral view. To date, tumor growth was only discovered in mice transplanted IH with 2 × 10⋀6 unselected MM PBMC from a fresh BM biopsy as early as 10 week post-transplantation. FACS analysis of these mice demonstrated successful engraftment with the presence of CD45+, CD19+ and CD138+ population in tumor, bone marrow, spleen and liver. In addition, expression of clonal light chain restriction in myeloma cells confirmed myeloma engraftment. Future studies will focus on expression of genes involved in sonic hedgehog pathway as analyzed by PCR to confirm the self-renewal capacity. Moreover, investigations on the effect of B cell-activating factor (BAFF) in BM microenvironment by transplanting MM CSC into BAFFxRG-KO mice are in progress. Disclosures: Jamieson: Bristol-Meyers Squibb: Research Funding.

Multiple Myeloma Cancer Stem Cell Animal Model.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1848-1848
Author(s):  
Christina C.N. Wu ◽  
Daniel Jacob Goff ◽  
Wenxue Ma ◽  
Heather Leu ◽  
Thomas A. Lane ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
B Cells ◽  
Plasma Cells ◽  
Memory B Cells ◽  
The Self ◽  
High Dose ◽  
Self Renewal ◽  
Immunomagnetic Bead

Abstract Abstract 1848 Poster Board I-874 Multiple myeloma (MM) is the second most common hematologic malignancy and characterized by clonal proliferation of CD138+ bone marrow plasma cells. Despite various treatment options few patients with MM have been cured. Furthermore, high relapse rates and recent evidence from xenogeneic transplantation models and primary MM marrow samples indicate that a rare population of cells or MM cancer stem cells (MM CSCs) within the marrow regenerates itself and may be responsible for drug resistance. These MM CSCs are phenotypically similar to memory B cells (CD138- CD34-CD19+) but differ in that they have the capacity to regenerate themselves or self-renewal. However, most of the reports on MM CSC animal models are established in NOD/SCID mice that require a larger number (1 – 10 × 106) of bead sorted cells for each animal. In addition, the latency of MM induction (4 – 6 months) in NOD/SCID mouse models and lack of in vivo tracking of the malignant clone preclude robust pre-clinical testing of novel therapeutic strategies that target MM CSC. Mononuclear cells were isolated from autologous mobilized peripheral blood of at least four primary MM patients after Ficoll gradient centrifugation followed by immunomagnetic bead depletion of CD34+ and CD138+ cells and/or further sorted using a FACSAria. The CD138-CD34- population was transduced with lentiviral luciferase GFP (GLF) and transplanted (10,000 to 106 cells per mouse) intrahepatically into neonatal RAG2-/- gamma chain-/- (RAG2-/-gc-/-) mice. Engraftment was compared to mice transplanted with either CD34+ or CD138+ cells. Mice were imaged with an in vivo imaging system (IVIS) to detect bioluminescent engraftment. Results showed that a relatively rare CD138- CD27+ population, resembling memory B cells (∼1.2%), persists in MM autografts and can engraft immunocompromised mice more rapidly and effectively than the CD138+ (Lin+) population of mature plasma cells. This data supports the persistence of CSCs despite high dose chemotherapy further underscoring the need for CSC targeted therapy. Bioluminescence was detected in live mice transplanted with as little as 60,000 cells of CD138- CD34- population and as soon as 4 weeks after transplantation. FACS analysis of these mice demonstrated successful engraftment with the presence of CD45+ and CD138+ population in bone marrow, spleen and liver and bioluminescence was also detected in the secondary transplantation of cells from MMCSC primary engraftment demonstrating the self-renewal capacity of this rare CD138- CD27+ population. Our results suggest that by utilizing a lentiviral GFP-luciferase system in a highly immunocompromised mouse strain fewer cells will be required to monitor MM engraftment and perhaps hasten disease development. Further studies to confirm the expression of selected IgG genes from myeloma cells and to characterize the self-renewal capacity with genes involved in developmental signaling such as sonic hedgehog and wnt pathways are underway. Disclosures: Goff: Coronado Biosciences: Research Funding.

Loss of Ezh2 Promotes the Development of Mutant-RUNX1 Induced MDS

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 402-402
Author(s):  
Goro Sashida ◽  
Satomi Tanaka ◽  
Makiko Mochizuki-Kashio ◽  
Atsunori Saraya ◽  
Tomoya Muto ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Suppressor ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
The Self ◽  
Post Transplantation ◽  
Self Renewal ◽  
Myeloid Malignancies ◽  
Empty Vector ◽  
Marrow Cells

Abstract Abstract 402 Polycomb group proteins are transcriptional repressors that epigenetically regulate transcription via histone modifications. There are two major polycomb-complexes, the Polycomb Repressive Complexes 1 and 2 (PRC1, PRC2). PRC2 contains SUZ12, EED, and EZH1/EZH2, and catalyzes the trimethylation of histone H3 at lysine 27 (H3K27me3), silencing target-genes. We have shown that the self-renewal of Ezh2-deficient HSCs is not compromised and H3K27me3 marks are not completely depleted in the absence of Ezh2, possibly as a result of Ezh1 complementation. EZH2 is generally thought to act as an oncogene in lymphoma and solid tumors by silencing tumor suppressor genes. Recently however, loss-of-function mutations of EZH2 have been found in myeloid malignancies such as AML, MDS and MPN, suggesting that EZH2 also functions as a tumor suppressor, although it remains unclear how EZH2 prevents the transformation of myeloid malignancies. RUNX1 is a critical transcription factor in the regulation of the self-renewal and differentiation of HSCs. RUNX1 mutations are frequently found in MDS, AML following MDS (MDS/AML) and de novo AML patients. One of the most frequent mutations, RUNX1S291fs, lacks the transactivation domain in C-terminus, but retains the RUNT DNA biding domain, resulting in a dominant negative phenotype. RUNX1S291fs-transduced bone marrow cells have been shown to generate MDS/AML in vivo. Given that RUNX1 and EZH2 mutations coexist in MDS and AML patients as reported recently, we generated a novel mouse model of MDS utilizing RUNX1S291fs retrovirus and Ezh2 conditional knockout mice in order to understand how EZH2 loss contributes to the pathogenesis of MDS upon genetic mutation of RUNX1. We first harvested CD34-Lin-Sca1+c-Kit+(LSK) HSCs from tamoxifen-inducible Cre-ERT;Ezh2wild/wild (EW) and Cre-ERT;Ezh2flox/flox (EF) mice (CD45.2) and transduced these cells with RUNX1S291fs retrovirus or an empty vector, which contains IRES-GFP. Then, we transplanted RUNX1S291fs-transduced Cre-ERT;Ezh2wild/wild (S291EW) or Cre-ERT;Ezh2flox/flox (S291EF) HSCs into lethally irradiated recipient mice (CD45.1) together with life saving dose 1×105 CD45.1 bone marrow cells. At 6 weeks post transplantation, we deleted Ezh2 via administration of tamoxifen, and observed disease progression until 12 months post transplantation. The empty vector transduced control mice with or without Ezh2 (EW and EF) did not develop myeloid malignancies. Two out of 16 S291EW mice died due to MDS progression, while 12 out of 16 and 1 out of 17 S291EF mice developed MDS and MDS/AML, respectively. S291EF mice showed significantly shorter median survival than S291EW mice (314 days versus undefined, p=0.037). In the peripheral blood, we observed significantly lower CD45.2+GFP+ chimerism in S291EF mice; however S291EF mice eventually showed macrocytic anemia and variable white blood cell counts accompanied with dysplastic features of MDS. Despite low CD45.2+GFP+ chimerism in peripheral blood, S291EF mice showed a higher chimerism of CD45.2+GFP+ cells in the bone marrow and had a significantly increased number of LSK and CD34-LSK cells compared to EW, EF, and S291EW mice, indicating that Ezh2 loss promoted HSCs/progenitors expansion, but impaired myeloid differentiation in the presence of RUNX1S291fs. We also saw enhanced apoptosis of CD71+Ter119+ erythroblasts in S291EF MDS mice, which may account for the anemia we observed. Since S291EF MDS bone marrow cells were transplantable in secondary experiments, we performed limiting-dilution assays to evaluate the frequency of MDS initiating cells and found that the frequency of MDS initiating cells was much higher in S291EF pre-MDS Lin-Mac1-Kit+ cells compared to S291EW pre-MDS Lin-Mac1-Kit+ cells. To understand this molecular mechanism, we performed gene expression analysis during MDS progression. S291EF MDS LSKs showed 1979 and 1875 dysregulated (>5-fold) genes, compared to EW LSK and S291EF pre-MDS LSK, respectively. We are now working to understand how these dysregulated genes are involved in the development of RUNX1S291fs-induced MDS after deletion of Ezh2. In summary, we have successfully recapitulated the clinical feature of MDS in mice reconstituted with Ezh2 null HSCs expressing a RUNX1 mutant, and demonstrated that Ezh2 functions as a tumor suppressor in this context. Disclosures: No relevant conflicts of interest to declare.

Bioluminescent Imaging of Human Leukemic Stem Cell Engraftment.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 696-696
Author(s):  
Catriona Jamieson ◽  
Mobin Karimi ◽  
Remi Creusot ◽  
Robert Negrin ◽  
Jason Gotlib ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Imaging System ◽  
Blast Crisis ◽  
Bioluminescent Imaging ◽  
Beta Catenin ◽  
Self Renewal ◽  
Advanced Phase

Abstract Previously, we found that candidate leukemic stem cells (LSC) involved in chronic myelogenous leukemic (CML) progression to myeloid blast crisis (BC) shared phenotypic characteristics with granulocyte-macrophage progenitors (GMP). However, CML GMP had activated a key self-renewal gene - beta-catenin. Aberrant in vitro self-renewal capacity could be specifically inhibited with axin - a potent beta-catenin antagonist (Jamieson et al, New Engl J Med2004;351:657–67). In order to determine whether these candidate LSC had enhanced in vivo self-renewal potential, we FACS-sorted hematopoietic stem cells (HSC), common myeloid progenitors (CMP), GMP, megakaryocyte-erythroid progenitors (MEP), CD34+CD38+ cells and blasts (lineage-positive cells) from advanced phase CML versus normal bone marrow or cord blood and transplanted them intrahepatically into newborn T, B and NK cell deficient (RAG2−−-γ−/−) mice (Traggiai et al. Science2004;304:104–7). Engraftment of human (ge;1%) CD45, CD19, CD3, and CD14-positive cells in the hematopoietic organs including bone marrow, liver, spleen and thymus of recipient animals was analyzed by FACS and compared with non-transplanted controls. In seven transplantation experiments performed with normal cord blood or bone marrow (n=24 mice), populations enriched for HSC, showed evidence of long-term engraftment, while committed progenitors including GMP did not. Conversely, in six experiments with myeloid BC CML (n=28 mice), GMP gave rise to long-term engraftment (7 of 11 mice) more frequently than HSC (2 of 6 mice) and blasts seldom engrafted (2 of 7 mice). These results suggested that LSC were enriched within the GMP fraction of myeloid BC CML. Subsequently, bioluminescent imaging (IVIS 100) was employed in order to track the kinetics of normal versus LSC engraftment more precisely. In 7 experiments involving normal marrow or cord blood (n=28 mice) and 3 experiments with advanced phase CML (n=18 mice), HSC, progenitor and blast (Lin+) populations were transduced with a lentiviral luciferase GFP vector and transplanted intrahepatically into newborn RAG2−/−γ−/− mice. Engraftment was monitored by weekly bioluminescent imaging as well as by tail vein bleeds to detect GFP expression. When mice were sacrificed, human engraftment in the liver, spleen, bone marrow and thymus was assessed by FACS analysis and sorted human CD45+ cells were transplanted into secondary recipients (n=4 experiments). In primary bioluminescent transplantation studies, CML HSC, CMP and GMP engrafted. Normal HSC demonstrated serial engraftment potential while more committed normal progenitors such as CMP, GMP and MEP did not. In contrast, CML blast crisis GMP demonstrated serial (2o and 3o) engraftment potential suggesting that they had gained the capacity to self-renew in vivo and thus, behaved like LSC. Hence, bioluminescent imaging of LSC engraftment in a highly immunocompromised mouse model can be used to detect LSC and may be utilized for pre-clinical evaluation of the effects of molecularly targeted therapy on LSC. Figure 1. Bioluminescent imaging was performed with the aid of a Xenogen™. IVIS 100 imaging system at 9 weeks post-transplant. Upper: RAG2−/γ0−/− mouse transplanted with no cells. Lower: Bioluminescence of 2° human CD45+GFP+ cells derived from mice transplanted with CML blast crisis GMP (mouse 1) or normal HSC (mouse 2) were compared with mice transplanted with primary normal Figure 1. Bioluminescent imaging was performed with the aid of a Xenogen™. IVIS 100 imaging system at 9 weeks post-transplant. Upper: RAG2−/γ0−/− mouse transplanted with no cells. Lower: Bioluminescence of 2° human CD45+GFP+ cells derived from mice transplanted with CML blast crisis GMP (mouse 1) or normal HSC (mouse 2) were compared with mice transplanted with primary normal

The TGF- β Family Member Growth Differentiation Factor 15 (GDF15) Regulates the Self-Renewal of Multiple Myeloma Cancer Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2954-2954
Author(s):  
Toshihiko Tanno ◽  
Akil Merchant ◽  
Jasmin R. Agarwal ◽  
Qiuju Wang ◽  
William Matsui
Keyword(s):  
Stem Cells ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cancer Stem Cells ◽  
The Self ◽  
Disease Stage ◽  
Dependent Manner ◽  
Self Renewal ◽  
Growth Differentiation Factor 15 ◽  
Differentiation Factor

Abstract Abstract 2954 Multiple myeloma (MM) cancer stem cells (CSCs) possess both enhanced tumorigenic potential and relative drug resistance suggesting they play a major role in disease relapse and progression. Therefore, a better understanding of the processes regulating MM CSCs may lead to the development of novel therapies that prevent tumor regrowth and improve long-term outcomes. Normal stem cells are tightly regulated by factors within the local microenvironment that include both soluble factors and direct contact with accessory cells. However, external factors regulating MM CSCs have not been identified. Recent studies have demonstrated that stromal cells in the MM bone marrow microenvironment secrete growth differentiation factor 15 (GDF15), a member of the TGF-b family. We initially studied the role of this cytokine in the pathogenesis of MM by examining circulating GDF15 levels in MM patients. Compared to healthy volunteers, we found that median GDF15 levels were significantly increased in MM patients (821 vs. 390 pg/ml; n=16; p<0.05) and increased with disease stage (Stage II=585 pg/ml, Stage III=1, 004 pg/ml). To examine the functional effects of GDF15 on MM cells, we cultured human MM cell lines (NCI-H929, RPMI 8226) with recombinant GDF15 and found that it induced the expansion of isolated CD138neg MM CSCs in a dose-dependent manner but had little impact on the growth of CD138+ plasma cells (Fig). Furthermore, GDF15 enhanced clonogenic myeloma growth as evidenced by increased colony formation that was maintained during serial replating, a surrogate for self-renewal. This effect appeared to be GDF15 specific since it could be blocked using anti-GDF15 antibody. Similarly, GDF15 treatment increased the in vitro clonogenic growth of MM CSCs from primary clinical bone marrow specimens. We also investigated the down-stream cellular pathways potentially mediating the effects of GDF15 and found that it activates the AKT signaling pathway known to improve the self-renewal of embryonic (ES) and normal hematopoietic stem cells. GDF15 also induced expression of the SOX2 transcription factor known to be upregulated in CD138neg MM CSCs. Since SOX2 is required for the self-renewal of ES cells and the generation of induced pluripotent stem (iPS) cells, its induction by GDF15 may also increase the self-renewal of MM CSCs. GDF15 is the first soluble factor identified that regulates MM CSCs, and its effects are mediated by the activation of highly conserved self-renewal programs. Disclosures: No relevant conflicts of interest to declare.

Regulation of Oxidative Stress by ATM Is Required for the Self-Renewal of Haematopoietic Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 369-369 ◽  
Author(s):  
Keisuke Ito ◽  
Atsushi Hirao ◽  
Fumio Arai ◽  
Sahoko Matsuoka ◽  
Tak W. Mak ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Bone Marrow ◽  
Stem Cell ◽  
Stromal Cells ◽  
Premature Aging ◽  
The Self ◽  
Self Renewal ◽  
Cell Functions ◽  
Damage Stability

Abstract It has been hypothesized that a signaling pathway for regulating aging and longevity may be involved in stem cell functions. To address the hypothesis we investigate roles of molecules that regulate aging on self-renewal capacity of HSC. Ataxia telangiectasia (A-T) is an autosomal recessive disorder caused by mutational inactivation of the ATM. ATM has a central role in maintenance of genomic stability via regulating cell cycle checkpoint in response to DNA damage, stability of telomere and oxidative stress. A-T patients display variety of sympotoms including high incidence of lymphoma and premature aging. In this study we investigated the effects of ATM deficiency on the hematopoietic system. ATM−/− mice have normal numbers of peripheral blood cells and colony-forming cells in the bone marrow (BM) at the age of 8 weeks. A frequency of KSL in the ATM−/− BM was similar to that of wild-type mice. However, the number of colony-forming cells derived from ATM−/− KSL cells after 6 weeks co-culture of stromal cells was significantly decreased. To directly assess a repopulating ability of the HSC in vivo, we performed a competitive reconstitution assay with congenic mice. Short-term (4-weeks) repopulation was not affected. However, there were dramatically fewer hematopoietic cells derived from ATM−/− BM at 16 weeks post-transplant, indicating that ATM has an essential role in the self-renewal of adult HSCs, but is not required for the differentiation or proliferation of hematopoietic progenitor cells. We next evaluated the effects of ATM deficiency on hematopoiesis in older mice. All ATM−/− mice older than 20 weeks exhibited a progressive pancytopenia with hypocellularity in bone marrow. The numbers of myeloid and erythroid precursors among ATM−/− BM MNCs were markedly decreased, KSL cells had disappeared, and co-culture on stromal cells showed that ATM−/− cells were no longer able to form any colonies after 2 weeks of culture. Taken together, our data indicate that chronic ATM deficiency in vivo results in progressive multi-lineage BM failure due to defective maintenance of the adult HSC pool. ATM is involved in oxidative defense, and the loss of ATM results in oxidative damage in several tissues. To elucidate the mechanism underlying the regulation of the HSC pool by ATM, we next evaluated ROS generation in HSC, and were able to demonstrate that the intracellular concentration of radical oxygen was higher in KSL cells from ATM−/− mice than from WT animals. Two members of CDK inhibitors, p16 and p19, were highly elevated in ATM−/− KSL cells after 2 days in vitro incubation with cytokines. Treatment with the permeant thiol N-acetylcysteine (NAC) abrogated the upregulation of p16 and p19 expression and BM failure corresponding to decrease in the level of intracellular ROS. The numbers of colonies formed from ATM−/− HSCs were restored to near-WT levels by treatment with either NAC or catalase in long term culture. Furthermore we found that NAC treatment of ATM−/− mice dramatically restored the repopulation capacity comparable to that of the WT. We conclude that self-renewal capacity of HSCs depends on ATM-mediated inhibition of oxidative stress. Our data support a model in which master regulator molecules govern the disparate processes of stem cell self-renewal, normal aging and tumor development.

Expression of a HoxC4 Retroviral Vector Mediates Expansion of Murine Hematopoietic Stem Cells with Normal Hematopoiesis in Transplanted Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2788-2788
Author(s):  
Lilia Stepanova ◽  
Brian P. Sorrentino
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Retroviral Vector ◽  
Post Transplantation ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow

Abstract Homeobox (Hox) transcription factors are important regulators of hematopoietic cell proliferation and differentiation. Of them, HoxB4 is of particular interest because overexpression promotes rapid expansion of mouse hematopoietic stem cells (HSCs) without causing neoplastic transformation. Despite the effects of HoxB4 overexpression on HSCs, mice that are homozygous for HoxB4 gene deletion have only subtle defects in HSCs and progenitor cells. We hypothesized that other paralogs of HoxB4 may also be capable of inducing HSC expansion could thereby compensate for loss of HoxB4 function. To test this hypothesis, we have studied the effects of retroviral overexpression of a HoxC4 gene in murine progenitors and HSCs. The murine HoxC4 cDNA was cloned and inserted into an MSCV vector that co-expresses an IRES-YFP reporter gene. We transduced murine bone marrow cells with a MSCV-HoxC4-YFP vector and compared the secondary replating efficiency of myeloid colonies (CFU-Cs) to that seen using either a MSCV-HoxB4-GFP or an MSCV-GFP vector. This assay tests for progenitor cell self-renewal which is increased using HoxB4-expressing vectors. Cells transduced with the MSCV-HoxC4-YFP vector formed 20–40 times more secondary CFU-Cs than with cells transduced with the MSCV-GFP control vector. This increase in CFU-C replating efficiency was equivalent to that seen with the MSCV-HoxB4-IRES-GFP vector. To test the in vivo effects of the MSCV-HoxC4-YFP vector on self-renewal of HSCs, we transplanted lethally irradiated mice with a mixture of cells; 20% transduced with the MSCV-HoxC4-YFP vector and 80 % mock-transduced. Peripheral blood analysis of the transplanted recipients up to 28 weeks post-transplantation showed that the percentage of cells transduced with the MSCV-HoxC4-YFP vector was 70–85% in both lymphoid and myeloid cells in the peripheral blood. A similar degree of chimerism was noted in concurrent controls using the MSCV-HoxB4-GFP vector. In contrast, the percentages of peripheral blood cells transduced with the MSCV-GFP vector was only 15–25%, paralleling the input ratios of transplanted cells. Secondary transplantation experiments showed stable levels of chimerism in both HoxC4 and HoxB4 groups, indicating that the expansion seen with the MSCV-HoxC4-YFP vector occurred at the HSC level. These results indicate that retroviral-mediated expression of HoxC4, like HoxB4, can cause significant expansion of HSCs in vivo. Because several other Hox genes can cause hematopoietic abnormalities and leukemia when expressed from a retroviral vector, we transplanted lethally irradiated mice with 4x106 cells that were transduced with the MSCV-HoxC4-YFP vector and monitored the animals for survival and complete blood counts. Now, at 33 weeks post transplantation, no tumor formation was observed in mice expressing either the HoxB4 or the HoxC4 vector, and peripheral blood counts have remained normal. Our results show that retroviral overexpression of HoxC4 can induce a significant expansion of the HSCs in vivo, and suggest that expression of HoxC4 may compensate for the loss of HoxB4 in knockout mice. We are currently analyzing the effects of HoxA4 and HoxD4 to determine if they share the same functional characteristics, and are also determining whether HoxB4 and HoxC4 are modulating the same downstream genes using microarray analysis of transduced murine bone marrow cells.

Differential Expression of c-Kit Identifies Hematopoietic Stem Cells with Variable Self-Renewal Potential.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2325-2325
Author(s):  
Joseph Yusup Shin ◽  
Wenhuo Hu ◽  
Christopher Y. Park
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Differential Expression ◽  
Peripheral Blood ◽  
Post Transplantation ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Abstract 2325 Hematopoietic stem cells (HSC) can be identified on the basis of differential cell surface protein expression, such that 10 out of 13 purified HSC (Lin−c-Kit+Sca-1+CD150+CD34−FLK2−) exhibit long-term reconstitution potential in single-cell transplants. HSCs express c-Kit, and interactions between c-Kit and its ligand, stem cell factor, have been shown to be critical for HSC self-renewal; however, HSCs express a log-fold variation in c-Kit levels. We hypothesized that differing levels of c-Kit expression on HSCs may identify functionally distinct classes of HSCs. Thus, we measured the function and cellular characteristics of c-Kithi HSCs and c-Kitlo HSCs (defined as the top 30% and bottom 30% of c-Kit expressors, respectively), including colony formation, cell cycle status, lineage fates, and serial engraftment potential. In methylcellulose colony assays, c-Kithi HSCs formed 5-fold more colonies than c-Kitlo HSCs (P=0.01), as well as 4-fold more megakaryocyte colonies in vitro. c-Kithi HSC were 2.4-fold enriched for cycling cells (G2-S-M) in comparison to c-Kitlo HSC as assessed by flow cytometry in vivo (15.4% versus 6.4%, P=0.001). Lethally irradiated mice competitively transplanted with 400 c-Kitlo HSCs and 300,000 competitor bone marrow cells exhibited increasing levels of donor chimerism, peaking at a mean of 80% peripheral blood CD45 chimerism by 16 weeks post-transplantation, whereas mice transplanted with c-Kithi HSCs reached a mean of 20% chimerism (p<0.00015). Evaluation of the bone marrow revealed an increase in HSC chimerism from 23% to 44% in mice injected with c-Kitlo HSCs from weeks 7 to 18, while HSC chimerism decreased from 18% to 3.0% in c-Kithi HSC-transplanted mice (P<0.00021). Levels of myeloid chimerism in the bone marrow and peripheral blood were not significantly different during the first 4 weeks following transplantation between mice transplanted with c-Kithi or c-Kitlo HSCs, and evaluation of HSC bone marrow lodging at 24 hours post-transplantation demonstrated no difference in the number of c-Kithi and c-Kitlo HSCs, indicating that differential homing is not the reason for the observed differences in long-term engraftment. Donor HSCs purified from mice transplanted with c-Kithi HSC maintained higher levels of c-Kit expression compared to those from mice injected with c-Kitlo HSC by week 18 post-transplantation (P=0.01). Secondary recipients serially transplanted with c-Kithi HSC exhibited a chimerism level of 40% to 3% from week 4 to 8 post-secondary transplant, whereas chimerism levels remained at 6% in mice injected with c-Kitlo HSC. These results indicate that c-Kithi HSCs exhibit reduced self-renewal capacity compared with c-Kitlo HSCs, and that the differences in c-Kithi and c-Kitlo HSC function are cell-intrinsic. Analysis of transplanted HSC fates revealed that c-Kithi HSCs produced two-fold more pre-megakaryocyte-erythroid progenitors and pluriploid megakaryocytes compared to their c-Kitlo counterparts in vivo, suggesting a megakaryocytic lineage bias in c-Kithi HSC. Consistent with this finding, the transplanted c-Kithi HSC gave rise to 10-fold more platelets and reached a maximum platelet output two days earlier than c-Kitlo HSC. To determine the potential mechanisms underlying the transition from c-Kitlo to c-Kithi HSCs, we assessed the activity of c-Cbl, an E3 ubiquitin ligase known to negatively regulate surface c-Kit expression in a Src-dependent manner. Flow cytometric analysis revealed 6-fold more activated c-Cbl in freshly purified c-Kitlo HSC compared to c-Kithi HSC (P=0.02), suggesting that functional loss of c-Cbl increases c-Kit expression on c-Kitlo HSCs. Mice treated for nine days with Src inhibitors, which inhibit c-Cbl activity, experienced a 1.5-fold and 2-fold increase in the absolute number of c-Kithi HSCs (P=0.067) and megakaryocyte progenitors (P=0.002), respectively. Thus, c-Cbl loss likely promotes the generation of c-Kithi HSCs. In summary, differential expression of c-Kit identifies HSC with distinct functional attributes with c-Kithi HSC exhibiting increased cell cycling, megakaryocyte lineage bias, decreased self-renewal capacity, and decreased c-Cbl activity. Since c-Kitlo HSC represent a population of cells enriched for long-term self-renewal capacity, characterization of this cell population provides an opportunity to better understand the mechanisms that regulate HSC function. Disclosures: No relevant conflicts of interest to declare.

Targeting the Hedgehog Signaling Pathway By PF-04449913 Limits the Self-Renewal of MDS-Derived Induced Potent Stem Cells (iPSC): Molecular Mechanisms

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 791-791
Author(s):  
Tetsuzo Tauchi ◽  
Seiichi Okabe ◽  
Seiichiro Katagiri ◽  
Yuko Tanaka ◽  
Kaoru Tohyama ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Research Funding ◽  
Molecular Mechanisms ◽  
Mononuclear Cells ◽  
Scid Mice ◽  
The Self ◽  
Bone Marrow Mononuclear Cells ◽  
Self Renewal

Abstract Background: Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders characterized by no efficient hematopoiesis and frequent progression to acute myeloid leukemia (AML). Even in low risk MDS, clonal hematopoiesis already dominates at diagnosis, and clones found in secondary AML originate from the MDS stage of disease, highlighting the need to specifically target the MDS-initiating clone. PF-0449913 is a potent and selective hedgehog pathway inhibitor that act by binding Smoothened (SMO) and blocking signal transduction. In xenograft models of human coloirectal and pancreatic cancer, treatment with PF-04449913 in combination with other anticancer agents reduced the tumor growth. Furthermore, PF-04449913 demonstrated preliminary antitumor activity in a phase I trial, when given as monotherapy in patients with several hematopoietic malignancy. In the present study, we investigated the molecular mechanisms by which PF-04449913 regulate the self-renewal of MDS-derived iPS cells (iPSCs) in vivo. Methods: We generated iPSCs from bone marrow mononuclear cells of two MDS patients (RAEB1 and RAEB2 by WHO clssification) with chromosome 5 deletion and complex karyotypic abnormalities, respectivly. Karyotyping analysis revealed that MDS-derived iPSCs have identical abnormalities to primary MDS cells. We also generated iPSCs from bone marrow mononuclear cells of normal volunteer as control. To investigate the effects of PF-04449913 on self-renewal and the relevance as a therapeutic target in MDS initiating cells, we examined the activity of PF-04449913 against MDS-derived iPSCs transferred NOD/SCID mice in vivo. NOD/SCID mice were injected sucutaneously with MDS-derived iPSCs or normal iPSCs then treated with PF-04449913 (100 mg/kg; p.o.) from day 10 for 28 days. We also used MDS-L, a myelodysplastic cell line establised from MDS patient with del(5q) and complex karyotypic abnormalities for in vitro studies. Results: Both MDS-derived iPSCs transferred NOD/SCID mice and normal iPSCs transferred NOD/SCID mice demonstrated the engraftment of CD34+CD38- positive cells by flow cytometry. However, the treatment with PF-04449913 reduced the population of CD34+CD38-positive cells in MDS-derived iPSCs transferred NOD/SCID mice. We isolated human CD45+ cells from the spleen of mice from each treatment group and injected equivalent numbers of CD45+ cells into secondary recipients. Following 50 days, all mice treated with vehicle engrafted with CD34+CD38- positive cells. In contrast, CD34+CD38-positive cells engraftment was not detected in recipient mice (n=3) from PF-04449913-treated donors. These results demonstrate the persistent effects of PF-0449913 on long term self-renewing MDS-initiating cells. We further examined the effects of Nanog pathway modulation on in vitro clonogenic growth. CD34+CD38- cells from MDS-derived iPSCs transferred NOD/SCID mice and MDS-L cells were treated with 2 mM of PF-04449913 for 72 hrs, washed free of drugs, and plated in quadruplicate in methylcellulose. At 14 days, colonies were counted as initial plating. The representative plate was then washed and cells were re-suspended and re-plated. After an additional 14 days, colonies were counted as secondary re-plating. Clonogenic recovery of untreated cells was normalized to 100% and plating results from all treatment groups were expressed as % control. PF-04449913 had only minimum effects on colony formation after initial plating over control cells. However, upon serial re-plating, secondary colony formations were significantly inhibited by PF-04449913 (p<0.001). To identify the mechanisms that limit the self-renewal of MDS-initiating ells by PF-04449913, NOD/SCID mice engrafted with CD34+CD38- fractions from MDS-derived iPSCs were treated with PF-04449913 (100 mg/kg; p.o.) for 14 days. PF-04449913 induced the expressions of p21Cip1, cleaved PARP and reduced the expression of BMI-1, c-Myc, Nanog, and Bcl-XL. Conclusion: Our preclinical results indicate that PF-04449913 have potential as an important option for controlling the drug-resistant MDS-initiating cells. It is expected that the PF-04449913 may become extremely useful therapeutic interventions in a number of hematological neoplasms, including MDS, where the persistence of cancer stem cells. Disclosures Ohyashiki: Sumitomo Dainippon: Membership on an entity's Board of Directors or advisory committees; Chugai Pharna KK: Research Funding; Bristol Meyer Squib KK: Research Funding; Jansen Pharma KK: Honoraria, Research Funding, Speakers Bureau; Celegen KK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis Pharma KK: Honoraria, Research Funding, Speakers Bureau; Kyowa Kirin KK: Honoraria; MSD KK: Honoraria; Nippo Shinyaku KK: Speakers Bureau; Toyama Kagaku KK: Speakers Bureau; Shinbaio Pharma KK: Honoraria; Asteras: Research Funding; Alexion Pharma KK: Research Funding; Teijin Pharma KK: Research Funding; Asahikasei: Research Funding; Taiho Yakuhin KK: Research Funding.

scholarly journals Hematopoietic Stem Cells Fail to Regenerate In Vivo Following Inflammatory Stress

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1472-1472
Author(s):  
Ruzhica Bogeska ◽  
Paul Kaschutnig ◽  
Stella Paffenholz ◽  
Julia Maassen ◽  
Jan-Philipp Mallm ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Research Funding ◽  
Post Transplantation ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Fold Reduction

Abstract An often-cited defining property of hematopoietic stem cells (HSCs) is their extensive or unlimited in vivo self-renewal capacity. We have recently described a novel mouse disease model forFanconi anemia, in which serial challenge with pro-inflammatory agonists that mimic infection, such aspolyinosinic:polycytidylic acid (pI:C), results in HSC attrition followed by a highly penetrant severe aplastic anemia, closely recapitulating the disease in patients (Walter et al., 2015, Nature). In order to explore the broader implications of these findings in the context of HSC self-renewal, we conducted apI:Cdose escalation regimen using standard C57BL6 mice. A single injection withpI:Cprovoked transient peripheral blood (PB)cytopenias, with the recovery of mature blood cell numbers correlating with HSCs being forced into active cell cycle. Injection with 1-3 rounds ofpI:C(1-3 x 8 injections) led to no discernable sustained impact on blood production as, at 5 weeks post-treatment, PB frequencies were in the normal range, as were the absolute numbers of HSCs and all progenitor compartments in the bone marrow (BM), as determined by flowcytometry. However, in vitro analysis of the proliferation and differentiation potential of 411 individual sorted long-term (LT)-HSCs 5 weeks after 3 rounds of pI:C challenge, revealed a decrease in the frequency of LT-HSCs able to generate progeny in vitro (1.6-fold reduction, p<0.05), and a 2-fold reduction in the total number of progeny produced per HSC, which was even more pronounced inmultilineage potential clones (2.6-fold decrease, p<0.0001) compared touni- or bi-lineage clones. In line with this data, competitive repopulation assays demonstrated a progressive depletion of functional HSC numbers with increasing rounds ofpI:C treatment, with a 1.8, 3.4 and 15.3-fold decrease in donorchimerism across all lineages at 6 months post-transplantation (p<0.01) following 1, 2 or 3 rounds ofpI:C treatment, respectively. Notably, robust engraftment (up to 65% donorchimerism, 6 months post-transplantation, p<0.01) was achieved when mice exposed to 3 rounds ofpI:C treatment were used as a recipient for non-treated BM cells in the absence of any irradiation conditioning, while engraftment was always <1% when non-treated controls were used as recipients. This excludes the possibility that the observed progressive depletion of functional HSCs was the result of artifacts associated with a compromised niche or the non-physiologic stress imposed on donor cells during transplantation. In order to test the kinetics of HSC recovery following HSC challenge, BM was harvested from mice at either 5, 10 or 20 weeks after treatment with 3 rounds of pI:C, and both competitive and limiting dilution transplantation assays (Table 1) were used to quantify HSC frequencies. Surprisingly, both assays demonstrated that HSCs failed to regenerate at all following pI:Cchallenge, directly contradicting the canonical view that HSCs possess extensive self-renewal capacity in vivo. The physiologic relevance of this observation was illustrated when we measured the hematologic parameters of aged mice that had been exposed to chronicpI:C treatment in early to mid-life. Although these mice had normal PB counts at 4 weeks post-treatment, at 2 years of age, peripheral bloodcytopenias and bone marrow aplasia became evident (Table 2), recapitulating clinically relevant features of non-malignant aged human hematopoiesis that are never seen in standard laboratory mice. Together, these data suggest that functional HSCs can be progressively and irreversibly depleted in response to environmental agonists, such as infection and inflammation, which force HSCs to reconstitute mature blood cells consumed by such stimuli. This model has clear implications relating to the role of adult stem cells in tissue maintenance and regeneration during ageing, and how stress agonists that are absent in most laboratory animal models, but would be ubiquitous in the wild, are likely key mediators of age-associated disease pathologies. Disclosures Frenette: PHD Biosciences: Research Funding; Pfizer: Consultancy; GSK: Research Funding.

scholarly journals Dnmt3a Is Essential for Hematopoietic Stem Cell Differentiation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 386-386 ◽  
Author(s):  
Grant A. Challen ◽  
Deqiang Sun ◽  
Mira Jeong ◽  
Min Luo ◽  
Jaroslav Jelinek ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Research Funding ◽  
Dna Methyltransferase ◽  
Donor Cell ◽  
The Self ◽  
Blood Cell Count ◽  
Serial Transfer ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Differentiation Capacity

Abstract Abstract 386 Aberrant genomic DNA methylation patterns are widely reported in human cancers but the prognostic value and pathological consequences of these marks remain uncertain. CpG methylation is catalyzed by a family of DNA methyltransferase enzymes comprised of three members – Dnmt1, Dnmt3a and Dnmt3b. Mutations in the de novo DNA methyltransferase enzyme DNMT3A have now been reported in over 20% of adult acute myeloid leukemia (AML) and 10–15% of myelodysplastic syndrome (MDS) patients. However, analysis of promoter methylation and gene expression in these patients has thus far failed to yield any mechanistic insight into the pathology of DNMT3A mutation-driven leukemia. In this study, we have used a conditional knockout mouse model to study the role of Dnmt3a in normal hematopoiesis. Hematopoietic stem cells (HSCs) from Mx1-Cre:Dnmt3afl/fl mice were serially transplanted into lethally irradiated recipient mice to study the effect of loss of Dnmt3a on HSC self-renewal and differentiation. We show that loss of Dnmt3a progressively impedes HSC differentiation over four-rounds of serial transplantation, while simultaneously expanding HSC numbers in the bone marrow. Examination of the bone marrow post-transplant revealed that control HSCs showed a gradual decline in their ability to regenerate the HSC pool at each successive round of transplantation, while in contrast Dnmt3a-KO HSCs show a remarkably robust capacity for amplification, generating 40,000 – 100,000 HSCs per mouse. Quantification of peripheral blood differentiation on a per HSC basis demonstrated in the absence of Dnmt3a, a cell division is more likely to result in a self-renewal rather than differentiation fate (Figure 1). Using semi-global reduced representation bisulfite sequencing (RRBS), we show that Dnmt3a-KO HSCs manifest both increased and decreased methylation at distinct loci, including dramatic CpG island hypermethylation. Global transcriptional analysis by microarray revealed that Dnmt3a-KO HSCs show upregulation of HSC multipotency genes coupled with simultaneous downregulation of early differentiation factors (e.g. Flt3, PU.1, Mef2c), likely inhibiting the initial stages of HSC differentiation. Upregulation of key HSC regulators including Runx1, Gata3 and Nr4a2 was associated with gene-body hypomethylation and activated chromatin marks (H3K4me3) in Dnmt3a-KO HSCs. Finally, we show that Dnmt3a-KO HSCs are unable to methylate and transcriptionally repress these key HSC multipotency genes in response to chemotherapeutic ablation of the hematopoietic system, leading to inefficient differentiation and manifesting hypomethylation and incomplete repression of HSC-specific genes in their limited differentiated progeny. In conclusion, we show that Dnmt3a plays a specific role in permitting HSC differentiation, as in its absence, phenotypically normal but impotent stem cells accumulate and differentiation capacity is progressively lost. This differentiation-deficit phenotype is reminiscent of Dnmt3a/Dnmt3b-null embryonic stem (ES) cells while markedly distinct from that of Dnmt1-KO HSCs which show premature HSC exhaustion and lymphoid-deficient differentiation, demonstrating distinct roles for the different DNA methyltransferase enzymes in HSCs. In light of the recently-identified DNMT3A mutations in AML and MDS patients, these studies are the first biological models linking mutation of Dnmt3a with inhibition of HSC differentiation which may be one of the first pathogenic steps occuring in such patients.Figure 1Dnmt3a-KO HSCs become biased towards self-renewal as opposed to differentiation. At each transplant round, the self-renewal quotient was calculated as the number of donor-derived HSCs recovered at the end of the transplant divided by 250 (the number of HSC initially transplanted). The differentiation quotient was calculated as (the white blood cell count per μl of blood at 16 weeks) X (percentage of donor-cell chimerism)/number of donor HSC at the end of the transplant. Over serial transfer, Dnmt3a-KO HSCs more rapidly lose their differentiation capacity compared to control HSCs, while sustaining robust self-renewal.Figure 1. Dnmt3a-KO HSCs become biased towards self-renewal as opposed to differentiation. At each transplant round, the self-renewal quotient was calculated as the number of donor-derived HSCs recovered at the end of the transplant divided by 250 (the number of HSC initially transplanted). The differentiation quotient was calculated as (the white blood cell count per μl of blood at 16 weeks) X (percentage of donor-cell chimerism)/number of donor HSC at the end of the transplant. Over serial transfer, Dnmt3a-KO HSCs more rapidly lose their differentiation capacity compared to control HSCs, while sustaining robust self-renewal. Disclosures: Issa: Novartis: Honoraria; GSK: Consultancy; SYNDAX: Consultancy; Merck: Research Funding; Eisai: Research Funding; Celgene: Research Funding; Celgene: Honoraria; J&J: Honoraria.

Sign in / Sign up

Export Citation Format

Share Document